In general, the present invention relates to the synthesis and use of hyaluronan (a.k.a., hyaluronic acid, sodium hyaluronate, or HA), and other hydrophilic polymers with pendant hydroxy groups that are not generally melt-processable in their ‘native’ state. As a naturally occurring polysaccharide with a large unbranched structure consisting of repeating disaccharides of N-acetylglucosamine and glucuronic acid, the structure of shown in FIG. 2A, present in vertebrate tissues and body fluids, HA has certain physical and biological properties, including viscoelasticity, hydrophilicity, lubricity and cell-activity regulation. Native HA and its currently available derivatives degrade before melting; thus, they cannot be thermally molded into custom shapes or otherwise thermally integrated with thermoplastic biomaterials. In biomechanical applications where characteristics such as maintaining a shape and/or supporting or protecting/cushioning a structure (e.g., joints, bone, cartilage and other tissue) are important, as well as for surgery instruments and aids, etc. and other mechanisms (whether biocompatibility is an issue)—degradation prior to melt point is an obstacle to using HA. The extreme hydrophilicity of HA does not permit use in conjunction with durable, hydrophobic biomaterials, such as polyethylene or polypropylene. Furthermore, the quick turnover within an animal body limits use of native HA in applications such as longer-term and permanent implants.
More-specifically, the instant invention is directed to a novel melt- or mold-processable hydrophilic polyanionic polymer with pendant hydroxy groups that are not generally melt processable in their native state, such as HA, as well as a method of synthesis of such a polymer. Of particular interest is hyaluronan/hyaluronic acid/sodium hyaluronate (generally, throughout referred to as “HA”); produced according to the invention, the melt- or mold-processable HA has a melting point below the point at which the polymer degrades. Pure (whether synthetic or genetically engineered, or native) HA has a melting point above the point of substantial degradation making it by-and-large impossible to mold or shape into structures suitable for use. A polymeric material produced according to the unique technique of the invention, provides a processable HA polymer that, once re-hardened (i.e., cooled or otherwise solidified), can be used in a variety of product applications, whether resultant structures are used ‘as is’ having been molded, extruded, or otherwise shaped into a mechanism/piece/device/etc. or employed as a member, component or subassembly of an assembly/system.
According to the invention a series of novel, melt- or mold-processable HA esters with varying aliphatic chain lengths are synthesized from silyl HA-quat. ammonium salt complex (preferably silyl HA-CTA, a silylated HA complex with cetyltrimethyl ammonium salt, a hyaluronan derivative). Introduction of aliphatic acyl groups (e.g., acid chlorides listed, TABLE 1) to HA disrupts the strong HA intermolecular bonding, reducing the crystallinity and producing appreciable thermoplasticization. Acylation takes place at the oxygen of the trimethylsilyloxy group —O—Si(CH3)3 in the silyl HA-CTA by removal of trimethylsilyl groups therefrom. Optionally, crosslinking may be performed during the shaping/molding of the HA esters into a structure/device, or thereafter, if at all. Native HA can then be regenerated/recovered by saponification/hydrolysis, removing acyl and -CTA groups. The structure/device of a preselected shape (e.g., porous or solid, bulk structure or fibers, etc.) may become a component of an assembly, a product that is further processed (e.g., seeded with cells, further shaped, etc.), integrated into another component (e.g., laminated, adhered, assembled, further shaped/molded together with a component, chemically-intermixed/intermingled, etc.), and so on.
Structure(s) produced according to the invention may be composed entirely of the melt- or mold-processable derivatized HA (or other polymer with hydroxy groups) and used alone, or used as scaffold for biological materials (e.g., cells, morphogenic proteins) or incorporated into a component, piece, module, feature, or mechanism/structure/member to produce a ‘system’ such that the HA's hydrophilic outer surface provided is interior- or exterior-facing, etc. A non-exhaustive list of possibilities contemplated for use of the derivative HA of the invention—including those where a generally hydrophilic outer surface is desirable—include: bearing surfaces or components for items such as gears, fishing rod eyelets, bearings of all types, joint and other weight-bearing mechanisms, whether incorporated as part of manufacturing equipment, as part of the manufactured product itself, etc.; flexible barrier surfaces separating a first and second area (such flexible barriers to include the membrane material or tubing used for catheter balloons, catheter tubing, hot air balloons, condoms, IV tubing, diaphragms, flexible bladders, etc.); transparent member surfaces including the transparent planar or curved polymeric films and sheet material used where optical clarity is sought, such as for fish tanks, polymeric covers for vehicle, water- or aircraft head-lamps and blinkers/fog-lights, covers for spot-lights, windows on or in a vehicle, aircraft, watercraft, and spacecraft, monitor and television screens, ophthalmic lenses, camera lenses and view-finders, etc.; in vivo implants of any of a variety of total or partial joint replacements, splints, stents, diaphragms, etc.; drag reduction surfaces and associated components of a vehicle, watercraft, aircraft and spacecraft such as hulls, pontoons, vehicle-body parts, blades/runners, etc., as well as the glide-surface of snowboards, water and snow skis, etc.; reaction resins for research or industrial components; topical dressing surfaces for dressings such as those used for medical/veterinary applications such as adhesive bandages, sterile pads for wounds and surgical procedures, bandage tape/adhesive, ace bandages, soft casts, etc.; and dental splints (to include mouth-guards, tooth/jaw-correction splints, etc.).
Further examples of applications for the invention include: tissue engineered scaffolds (porous, seeded with cells, etc.) for cartilage and other tissue repair and treatment, wound dressings, artificial skin, viscoelastics for intra-surgical protection and prevention of post-operative adhesions, hydrophilic, lubricious and/or anti-fouling and/or anti-coagulant coatings (e.g., catheters, contact lenses, dialysis membranes), drug release/delivery devices, and biodegradeable materials (e.g., nerve guides).
While the focus, here, is of HA (i.e., a hydrophilic polyanionic polymer with hydroxy groups that is not generally melt processable in currently available native or derivative state(s)) other biomaterials exhibiting similar characteristics that would benefit from molding or shaping are contemplated hereby as a starting polymer. Examples shown herein showcase the synthesis of unique HA esters, i.e., melt- or mold-processable HA derivatives having hydrophobicity and compatibility with other generally hydrophobic materials. Derivatization of HA according to the invention permits control of its hydrophobicity, expanding the range of useful solvents and non-solvents during synthesis and fabrication into end product. In the spirit and scope of design goals contemplated hereby, the novel melt- or mold-processed polymer produced according to the invention may be made by chemically modifying a wide variety of hydrophilic polyanionic polymers containing pendant hydroxy groups, including without limitation: polyanionic polyhydroxy polymers such as polysaccharides and glycoaminoglycans.
Hyaluronan was first isolated from bovine vitreous humor in acid form in 1934; it was coined “hyaluronic acid” meaning uronic acid from hyaloid (vitreous). The first non-inflammatory fraction of sodium hyaluronate, called NIF—NaHA, which was free of impurities that could cause inflammatory reactions was synthesized by Endre Balazs. HA has certain physical and biological properties: (1) Viscoelasticity. Hyaluronan carries one carboxyl group (—COOH) per disaccharide unit, which is dissociated at physiological pH thereby conferring a polyanionic characteristic to the compound; (2) Hydrophilicity. Hyaluronan acts as a water-retaining polymer network in many tissues; it can hold large amounts of water like a molecular sponge. The hydrodynamic volume of HA in solution is 1000 times larger than the space occupied by the unhydrated polysaccharide chain. (3) Lubricity. The extraordinary viscoelastic properties also make hyaluronan ideal as a lubricant. Hyaluronan in synovial fluid complexes with proteins and penetrates the surface of cartilage, forming a layer of HA protein complex that serves as a lubricating layer in joints and other tissue surfaces that slide along each other. Under slow mechanical loading it behaves as a viscous oil-like lubricant. At higher mechanical loading rates the HA layer becomes a highly deformable elastic system: it absorbs and converts an imposed stress into an elastic deformation, then rebounds to the original condition when the stress is removed.
Not only does HA act as a vital structural component of connective tissues, it also plays an role in diverse biological processes, such as cellular migration, mitosis, inflammation, cancer, angiogenesis and fertilization. However, HA's high solubility, rapid degradation and short residence time in water have historically limited biomedical application of naturally occurring HA, particularly in tissue engineering and viscoseparation applications. Four pendant groups on hyaluronan are available (FIG. 2A) for chemical modification: carboxyl, hydroxyl, acetamido group and the reducing end-group. Prior attempts of others at synthesizing HA derivatives have been directed at modifying one or more of these groups, resulting in much different derivative end-product (none have thermoplastic characteristics, as they by-and-large have no meltable endotherms).
Two groups of commercialized hyaluronan derivatives, include HYLAN and HYAFF®: HYLAN is a brand name for a crosslinked hyaluronan in which crosslinking only occurs on hydroxyl groups, not affecting carboxyl and acetamide groups, this cross-linking was done by Balazs and his colleagues. HYAFF® is the brand name of a class of hyaluronan esters with the free carboxyl group of glucuronic acid esterified using different types of alcohols (delta Valle et al., U.S. Pat. No. 4,851,521, 1989). patent application No. US 2002/0143171 A1 published 3 Oct. 2002 to Yui et al. focuses on a chemically modified HA for use as pharmaceuticals, foodstuffs, cosmetics (e.g., moisturizing agent, lotion) and similar flowable, gel-like substances, but their crosslinked polymer can not melt without first degrading. Yui et al. modified the hydroxyl groups of HA with an acid halide carrying photoreactive groups, such as cinnamoyl chloride, in DMF solution in the presence of pyridine. The product was dissolved in DMF, and was subjected to ultraviolet radiation to crosslink HA. The applicants hereof have published earlier work: US App. US 2003/0083433 A1 filed on behalf of the assignee hereof for the applicants on 29 Oct. 2002, and is hereby incorporated herein by reference to serve as technical background support.
‘Animal’ as used throughout includes any multicellular organism having a body that can move voluntarily and actively acquire food and digest it internally, including human beings and other mammals, birds and fish. ‘Extrude’/‘extrusion’ as used includes a molding technique as follows: the moldable material is forced through the shaping die of an extruder; may have a solid or hollow cross section. The following general acronyms, if used throughout, are decoded on the following page:    HA—Hyaluronic acid/hyaluronan/sodium hyaluronate.    HA-CPC—the complex of HA polyanion and cetylpyridinium salt.    HA-CTA—the complex of HA polyanion and cetyltrimethyl ammonium salt.    HA−-QN+—the complex of HA polyanion & long-chain paraffin ammonium cation.    HMDS—hexamethyldisilazane, a silylation agent.    THF—Tetrahydrofuran.    TMCS—trimethylchlorosilane, a silylation agent.    QN+—long-chain paraffin ammonium cation.